The long-term goal of this project is to elucidate the cellular mechanisms by which the 50-kDa ezrin-bind protein (EBP50) regulates parathyroid hormone receptor (PTHR)-mediated signaling and function in bone. Mice with targeted deletion of EBP50 exhibit a bone phenotype, as do patients with EBP50 mutations. Although it is thought that the bone disorder arises as a secondary consequence of renal dysfunction, our preliminary data identify direct effects of EBP50 on bone. This suggested a novel mechanism by which mutations interfere with EBP50 function and, by extension, that EBP50 is dynamically regulated by PTH in open and closed conformations. The unifying idea of the present proposal is that novel structural determinants in EBP50 and their posttranslational modification dictate EBP50 function on PTHR activity in bone. Three specific aims are developed to test this idea. In Aim 1 we will characterize EBP50 conformations and dimerization to test the hypothesis that the described mutations lock EBP50 in a closed configuration that interferes with PTHR function. These experiments will use molecular biological maneuvers to examine static interactions, molecular modeling to predict the effect of amino acid mutation on binding affinity, and biophysical measurements of fluorescence resonance energy transfer microscopy to acquire dynamic interactions in living cells and in real time, and surface plasmon resonance to quantify protein-protein interactions. Aim 2 will define post- translational modifications of EBP50 that determine its function. This will be accomplished by testing the hypothesis that PTH-induced phosphorylation of EBP50 induces the closed configuration. We will apply mass spectrometry to identify site-specific EBP50 phosphorylation, and molecular biological tools with phospho-mimics and phospho-resistant EBP50 derivatives to determine their structural conformation and their actions on bone cells. Aim 3 will delineate the direct effects of EBP50 on bone to test the hypothesis that EBP50 regulates bone development and turnover. Several approaches will be applied including allograft transplantation to determine if the bone phenotype of EBP50-null mice can be rescued by transplanting marrow stem cells from wild-type mice. Other experiments will involve transfecting bone cell models with mutant EBP50 or EBP50 harboring phospho-mimics or phospho- resistant forms of EBP50 to determine how these influence PTHR action. These studies will quantitatively examine the relations between EBP50 structure and function and characterize a novel mechanism to explain the regulation and origin of EBP50 effects on bone. The findings will generate new information that is relevant to understanding bone turnover. The outcomes will help define potential therapeutic targets for improved treatment of osteoporosis and other metabolic bone diseases.